We have continued to analyze the phenotype of single, double (DM), and triple (TM) mutants of myoB, myoC and myoD, the three classic myosin I isoforms identified to date in Dictyostelium. These three isoforms are closely related in terms of tail domain structure and their localizations in the cell largely overlap. All the mutants were characterized with regard to whole cell motility, streaming behavior, phagocytosis, doubling times, and endocytic flux (including uptake and efflux kinetics and transit time). We found for these three classic myosin I isoforms evidence of largely nonoverlapping functions (whole cell motility), overlapping functions that are nonadditive (phagocytosis), and overlapping functions that are more than additive (i.e. synergistic) (streaming, endocytosis). While some of the apparent lack of functional overlap could be due to differences in the actual amounts of these three isoforms in the cell, other data indicate that these isoforms have both overlapping and distinct functions. These studies identify a number of cellular processes (whole cell motility, phagocytosis, endocytosis) that are clearly affected to a very significant extent by the absence of these classic myosin I isoforms. The complete sequence of the Dictyostelium myo J heavy chain gene has been determined. The gene encodes a 2241-residue heavy chain polypeptide (MW 258 kDa) that is comprised of an N-terminal 944 -residue myosin head domain (that contains 5-6 IQ motifs), a central 863-residue alpha helical-coiled-coil domain (that contains six hinges), and a C- terminal 434-residue globular tail domain. This primary structure predicts a two-headed molecule that is incapable of self-assembly into filaments. While these properties are reminiscent of class V myosins, various sequence analyses indicate that myo J represents a new class within the myosin superfamily (XI). Myo J- cells have been generated by homologous recombination and are currently under analysis. Myo J has been localized by light immunofluorescence to the contractile vacuole using a new, high-titre, myo J- specific antibody. Current efforts are directed a cloning the gene for what is probably the only other myo J isoform in Dictyostelium. Creation and analysis of a double mutant should reveal the function of class XI myosins in vivo. New projects that involve the localization of vertebrate myosin V in various cell types and the analysis of the phenotypes of these same cell types isolated from myosin V- mice are underway.